Team:ETH Zurich/Decoder/Results

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TOP10 colonies were transformed with <partinfo>BBa_K137055</partinfo> , <partinfo>BBa_S04039</partinfo> and <partinfo>BBa_K909014</partinfo>. The transformed cells as well as the TOP10 WT were cultivated ON in falcon tubes containing 4 mL of LB. After normalization to an OD of 0.15, the cells were lysed and mixed with 6X Laemmli buffer.  
TOP10 colonies were transformed with <partinfo>BBa_K137055</partinfo> , <partinfo>BBa_S04039</partinfo> and <partinfo>BBa_K909014</partinfo>. The transformed cells as well as the TOP10 WT were cultivated ON in falcon tubes containing 4 mL of LB. After normalization to an OD of 0.15, the cells were lysed and mixed with 6X Laemmli buffer.  
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PabA has a size of 21,656 kDa (193 aa), PabB of 71,743 kDA (628 aa).
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PabA has a size of 21,656 kDa (193 aa), PabB/C of 71,743 kDA (628 aa).

Revision as of 21:01, 26 September 2012

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Navigate through Overview, Design and Results

Contents

Hybrid promoters

We succesfully cloned the 4 hybrid promoters. Results can be found on the partsregistry.

PABA generator

Cloning

Fig.: Plasmid map of <partinfo>BBa_K909014</partinfo> in pSB1C3.

We were succesful in constructing both parts <partinfo>BBa_K909014</partinfo> (2699bp) and <partinfo>BBa_K909015</partinfo> (2656bp) based on the already existing parts <partinfo>BBa_K137055</partinfo> and <partinfo>BBa_S04039</partinfo>. The vector backbone is <partinfo>pSB1C3</partinfo> (2079bp). They differ from each other due to the fact that <partinfo>BBa_K909014</partinfo> contains the constitutive promoter <partinfo>BBa_J23100</partinfo> with two NheI restriction sites.

To verify the sizes of the constructs, the plasmids containing <partinfo>BBa_K909014</partinfo> and <partinfo>BBa_K909015</partinfo> were digested with

1. NheI & PstI

  • Expected bands of <partinfo>BBa_K909014</partinfo>: 2685 bp & 2061 bp
  • Expected bands of <partinfo>BBa_K909015</partinfo>: 4724 bp

2. XbaI & PstI

  • Expected bands of <partinfo>BBa_K909014</partinfo>: 2721 bp & 2048 bp
  • Expected bands of <partinfo>BBa_K909015</partinfo>: 2678 bp & 2048 bp
Fig.:Plasmid map of <partinfo>BBa_K909015</partinfo> in pSB1C3.


Fig.1.: Digests left to right: Ladder, <partinfo>BBa_K909014</partinfo> (NheI/PstI), <partinfo>BBa_K909014</partinfo> (XbaI,PstI),<partinfo>BBa_K909015</partinfo> (NheI/PstI), <partinfo>BBa_K909015</partinfo> (XbaI,PstI).









Expression of the proteins PabA & PabB/C

TOP10 colonies were transformed with <partinfo>BBa_K137055</partinfo> , <partinfo>BBa_S04039</partinfo> and <partinfo>BBa_K909014</partinfo>. The transformed cells as well as the TOP10 WT were cultivated ON in falcon tubes containing 4 mL of LB. After normalization to an OD of 0.15, the cells were lysed and mixed with 6X Laemmli buffer.

PabA has a size of 21,656 kDa (193 aa), PabB/C of 71,743 kDA (628 aa).


SDS PAGE


In a further step <partinfo>BBa_K909015</partinfo> is joined to the PL hybrid promoter <partinfo>BBa_K909011</partinfo> for implementation.


Outlook

After assembling all parts for the decoder, we want to test the circuit by simulating blue and red light with aTc and IPTG. Our output system is based on fluorescent proteins (eYFP, mCherry and GFP) giving us the possibility to analyse the result by single cell analysis (FACS).

Figure: Boolean logic for testing of Decoder.


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